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Neuron Tracing and Reconstruction

Reconstruct and perform complex analyses of neurons
MBF Bioscience > Neuron Tracing and Reconstruction

What is Neuron Reconstruction and Why is It so Important?

Neuron reconstruction is the process of creating digital reconstructions of the entire (or sometimes only specific portions) of neurons. Neuron reconstruction includes delineating and reconstructing the axon, dendrites soma, and other sub-cellular components of a neuron, thereby creating a digital, geometric model of the neuron. Researchers use neuron reconstruction techniques for reconstruction and morphological analysis of neurons in tissue sections, tissue slabs, intact brains, and cell culture. Neuron reconstruction is typically performed using light microscopy imaging, sometimes it is performed using electron microscopy imaging. 

 

The resulting 3D reconstructions are used to visualize and analyze not only overall neuronal process length, but also other morphological quantities such as branching frequency, segment length, branch angle, spine or receptor density, and segment diameter. These digital reconstructions can be morphometrically analyzed to understand more about intricate neuronal structures. The reconstructions can also be used with electronic modeling software to model the electrical properties of neurons. The morphological structure and connectivity of somas, dendrites, axons, spines, varicosities and synapses provide extremely valuable information on the workings of neurons throughout the body, ranging from brain circuits to the peripheral nervous system.

 

Santiago Ramón y Cajal, was the first to perform neuron reconstruction. He did so with a pencil and paper, creating graphic representations of neurons he observed. Edmund Glaser, MBF Bioscience co-founder, and Hendrick van der Loos performed the first digital neuron reconstruction in the early 1960’s. Today, neuron reconstruction in performed in 1000’s of laboratories around the world using techniques that range from computer assisted, to fully-automatic. 

Fields Of Study

  • all major neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders
  • intellectual disability
  • traumatic brain injury
  • neuroAIDS
  • long COVID
  • ischemia
  • mild cognitive impairment
  • vascular dementia
  • Alzheimer’s disease
  • Parkinson’s disease
  • Huntington’s disease
  • schizophrenia
  • autism spectrum disorder
  • attention deficit hyperactivity syndrome
  • depression
  • drug addiction
  • neuroinflammation
  • multiple sclerosis
  • spinal cord injury
  • neuropathic pain
  • amyotrophic lateral sclerosis
  • epilepsy

Why Do Researchers Reconstruct Neurons?

An accurate digital reconstruction of neuron morphology is critical for the most accurate quantitative analyses. Neuron reconstruction is an important facet of researching in learning, memory, and behavior. It is also used in research into all major neurodevelopmental, neuropsychiatric, neurodegenerative and neurological disorders, including: intellectual disability, traumatic brain injury, neuroAIDS, ischemia, mild cognitive impairment, vascular dementia, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, epilepsy, schizophrenia, autism spectrum disorder, attention deficit hyperactivity syndrome, depression, drug addiction, neuroinflammation, multiple sclerosis, spinal cord injury  and neuropathic pain.

How Do You Reconstruct Neurons?

There are two general methods for reconstructing neurons. One popular method is to obtain images (2D or 3D) from a microscope and then use software that can import the image data and provide you with a set of tracing and segmentation tools. Some software may also have some built-in analyses tools. 

 

 Three important factors with this method are:

 

1. Image Data Quality: The XY and Z resolution are very important as well as high contrast and low noise in the images

 

2. Neuron Reconstruction Tools:  These tools can vary substantially from very basic drawing tools that come in free software to highly specialized tools that are designed specifically to reconstruct as accurate and detailed digital model of the neuron as possible

 

3. Analyses Tools: These tools can also very substantially from very basic measurement tools to highly sophisticated tools designed specifically to provide quantitative morphometric data that are relevant to neuroscience researchers 

A second popular method for reconstructing neurons does not require any previously acquired image data at all. This method requires a computer-controlled microscope-based system with software that provides neuron reconstruction tools to trace from a live camera image. This method is very popular for reconstructing Golgi stained neurons or other brightfield staining methods as there is no concern for tissue bleaching.

 

Three important factors with this method are:

 

1. Microscope Control: The software used must be capable of fully-controlling all motorized functions of the microscope to create one fully integrated system and the motorized stage must be off a high enough resolution in order to record accurate XYZ position data

 

2. Reconstruction Tools: The software must include neuron reconstruction tools that allow the user to trace over a live camera image and across multiple fields of view and even multiple tissue sections

 

3. Image Analysis: The software must provide similar tools for image analysis that would be used in the first method.

What Kind of Analysis I Can Obtain Using Neuron Reconstruction?

All the data produced in Neurolucida and Neurolucida 360 is stored in a published (non-proprietary), vector format. It is extremely easy to edit on 3D or represent and scale graphics for your publications. The data can be analyzed with Neurolucida Explorer (included with Neurolucida and Neurolucida 360). This is an extremely powerful and comprehensive quantitative analysis package with more than one hundred types of morphometric analyses. They comprise multi-level morphometric analysis (length, diameters or volumes from population to segment level), topology and branch connectivity (degree, vertex analysis), spatial analysis (i.e., Sholl, convex hull), orientation and region-specific analysis (polar histogram, closed surface). If you don’t know the most suitable analysis for your experiment, you can ask for help from our research support team which is composed of Ph.D. neuroscientists and experts in microscopy.

What Makes a Good, Accurate Reconstruction?

We’ve found that these are the most critical factors:

  • Accuracy of digitizing small discrete positions in all 3 axes, with proper calibration
  • Accurate thickness traced and determine for all axon and dendritic processes
  • Accurate soma reconstruction
  • All branching structures are traced and connected properly and nodes are identified
  • Branching structures can be segmented and color coded
  • Reconstruction matches the image data when viewed in 3D
  • Spines are identified and classified
  • Varicosities and boutons are identified
  • Neuron in serial sections are joined into a comprehensive digital reconstruction

What Makes Some Neuron Reconstructing Software Better Than Others?

Carefully compare solutions you review on these features:

  • Accuracy & validity of the reconstruction produced
  • Ease of use
  • Speed of tracing & reconstruction
  • Ability to detect, trace & reconstruct sub-celluar structures
  • Automatic trading modules: fully automatic, user guided, manual
  • Ability to handle big image data files
  • Ability to handle multiple neurons
  • Availability of technical support
  • Built-in morphometric analyses tools
  • Built-in tools for publishing data

What Should You Look for When Purchasing a Neuron Reconstructing System?

If you are looking for a microscope-based system you should check whether it is able to control the newest models of microscopes, motorized stages and cameras as well as command internal microscope components like light sources, filter wheels and motorized optics to automatize imaging. When you look for an offline system, it should be accurate and have its results validated. It should have a fast learning curve and yet be a powerful and flexible analysis tool with different algorithms and parameter configurations, so you can trace a wide range of labels and image types, while having the ability to perform the specific analyses you need for your research. Additionally, it is essential to have support for many different microscope image formats, so data can be quickly imported without the need for file format conversions that slow the process and often result in missing metadata.

An eight-channel fluorescence whole slide imaging and analysis system controlled by Neurolucida and built with a Zeiss AxioImager microscope with an ApoTome.

What is the Best Neuron Reconstructing Software?

Neurolucida and Neurolucida 360 are two different software applications that provide tools for 2D and 3D tracing dendrite, axons, spines, synapses, somas and varicosities. You can also use them to manually map the brain to give anatomical context to neuronal pathways, cells and synaptic distribution (for automatic brain nuclei mapping see NeuroInfo). 

What is Neuronal Tracing?

Neuronal tracing is a technique used in neuroscience to map the connections and pathways of neurons in the nervous system. This technique helps researchers understand neural circuits, connectivity patterns, and brain organization

 

 Here’s a brief overview of how it works:

 

    1. Tracer injection: A tracer substance is introduced into a specific area of the nervous system.

 

    1. Tracer types:

   Anterograde tracers: Move from the cell body to axon terminals

   Retrograde tracers: Travel from axon terminals back to the cell body

   Transneuronal tracers: Can cross synapses to label connected neurons

 

    1. Transport: The tracer is taken up by neurons and transported along their axons.

 

    1. Visualization: After a suitable time period, the tissue is processed and examined using various imaging techniques like brightfield, fluorescence, 2 photon, confocal or light sheet microscopy. The most common method of performing neuron tracing directly on a microscope system is using Neurolucida. When microscope imaging systems are used for 3D imaging, eg light sheet, confocal, etc, image stacks are traced and reconstructed with Neurolucida 360.

 

    1. Analysis: The traced neurons are morphometrically analyzed using Neurolucida Explorer.

 

Common tracers include:

 

    1. Fluorescent dyes:
        • DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)
        • DiO (3,3′-dioctadecyloxacarbocyanine perchlorate)
        • Fluoro-Gold
        • Fast Blue
        • Fluorescein isothiocyanate (FITC)
    2. Radioactive amino acids:
        • Tritiated amino acids (³H-amino acids) like ³H-leucine or ³H-proline
        • ¹⁴C-labeled amino acids
    3. Viral vectors:
        • Adeno-associated virus (AAV)
        • Rabies virus (RABV)
        • Herpes simplex virus (HSV)
        • Pseudorabies virus (PRV)
    4. Biotinylated dextran amines (BDA):
        • BDA-3k (3,000 molecular weight)
        • BDA-10k (10,000 molecular weight)
    5. Other common tracers:
        • Horseradish peroxidase (HRP)
        • Cholera toxin subunit B (CTB)
        • Phaseolus vulgaris-leucoagglutinin (PHA-L)

Each of these tracers has specific properties that make them suitable for different types of neuronal tracing studies. For instance, some are better for anterograde tracing, while others are more effective for retrograde tracing. The choice of tracer depends on factors like the specific neural pathways being studied, the desired visualization method, and the experimental timeframe.

 

What is Neuron Tracing?

Neuron tracing and reconstruction is a process used to create detailed digital representations of individual neurons. This technique is crucial for understanding neuronal morphology and connectivity. Here’s an overview of the process:

 1. Image Acquisition

 

High-resolution imaging techniques are used, such as:

      • Confocal microscopy
      • Two-photon microscopy
      • Brightfield microscopy

 

Systems typically include the capabilities to:

      • Acquire a series of 2D slices or z-stacks
      • Perform tracing directly on a microscope with Neurolucida controlling a camera, XYZ stage, focus encoder, illumination devices, etc.

 

2. Image Preprocessing

 

    • Noise reduction
    • Deconvolution
    • Alignment of image stacks

3. Segmentation
 

Identifying and separating the neuron from the background. 

4. Tracing

 

Following the neuron’s structure through the image stack. This can be done:

      • Manually
      • Semi-automatically
      • Fully automatically

 

Involves tracing:

      • Soma (cell body)
      • Dendrites
      • Axon
      • Spines and boutons (synaptic structures)

 

5. 3D Reconstruction

 

  • Combining traced elements into a 3D model
  • Using surface rendering or skeleton-based representations

 

 
 

7. Visualization

 

Creating 3D renderings for visual analysis and presentation.

 

6. Analysis

 

Quantifying morphological features like:

    • Branch lengths
    • Branch angles
    • Spine densities
    • Bouton distributions

 

Common Software Tools

 

Common software tools for neuron tracing and reconstruction include:

 

This process allows researchers to study neuronal structure in great detail, contributing to our understanding of brain organization, neuronal function, and how morphology relates to neural computation.

 

What is the Difference Between Neuronal Tracing and Neuron Tracing?

While the terms “neuronal tracing” and “neuron tracing” are often used interchangeably, there can be a subtle distinction between them in some contexts:

Neuronal tracing:

 

  • Typically refers to the broader technique of mapping neural pathways and connections in the nervous system.
  • Often involves studying populations of neurons or entire neural circuits.
  • Usually employs chemical tracers or viral vectors to visualize connections between different brain regions or groups of neurons.

 

Neuron tracing:

 

  • Can refer more specifically to the process of reconstructing the morphology of individual neurons.
  • Often involves detailed imaging and digital reconstruction of a single neuron’s structure, including its dendrites, axon, and cell body.
  • Frequently used in computational neuroscience and in creating detailed 3D models of neurons.

 

In practice, the distinction is not always clear-cut, and many researchers use these terms interchangeably. The choice of term might depend on:

 

  • Scale: “Neuron tracing” might be used for single-cell analysis, while “neuronal tracing” could imply a larger-scale study.
  • Technique: Some might use “neuron tracing” for computer-aided reconstruction techniques, reserving “neuronal tracing” for tracer-based methods.
  • Field of study: Certain subfields of neuroscience might prefer one term over the other.

 

Our Solutions for Neuron Tracing and Reconstruction

Neurolucida 360®

Neurolucida 360 is designed for automatic 3D and 2D reconstruction from acquired images from brightfield, fluorescence, confocal, and light sheet microscopes. Using multiple algorithms and a 3D interactive reconstruction environment, it can easily detect and trace structures automatically. It is compatible with different image modalities (confocal, 2-photon, lightsheet) and many proprietary image formats (.czi, .lif, .oib, .nd2, etc.). Neurolucida 360 is the most advanced automatic neuron reconstructing software. It can be used on 2D and 3D images obtained from a wide variety of specimens. Its capabilities for reconstructing neurons and modeling dendritic spines are unparalleled.

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Neurolucida®

Neurolucida is primarily a manual computer-assisted neuron reconstruction system that can be integrated directly with microscopes. In addition to reconstructing structures, it can be used as your primary image acquisition software package, so you can do brightfield and multichannel fluorescent stack imaging, multifocal tiling, image post-processing. Neurolucida is the most widely used neuron reconstruction system in the world due to its ability to accurately trace virtually any neuron in any specimen. It is the gold standard in neuron reconstruction.

 

 

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